Spectroscopy is a general term for the process of measuring energy or intensity as a function of wavelength in a beam of light or radiation. Many conventional spectroscopes, and components comprising a spectroscope system, also referred to as an instrument, may include basic features and components such as a slit and a collimator for producing a parallel beam of radiation, one or more prisms or gratings for dispersing radiation through differing angles of deviation based on wavelength, and apparatus for viewing dispersed radiation. Spectroscopy uses absorption, emission, or scattering of electromagnetic radiation by atoms, molecules or ions to qualitatively and quantitatively study physical properties and processes of matter.
Light or radiation directed at a target, or sample of physical matter, during operation of a spectroscope system may be referred to as incident radiation. Redirection of incident radiation following contact with a sample of physical matter ("sample") commonly is referred to as scattering of radiation. To the extent that atoms or molecules in a sample absorb all or a portion of incident radiation, rather than reflect incident radiation, a sample may become excited, and the energy level of the sample may be increased to a higher energy level. Electromagnetic radiation, including incident radiation, that passes through a sample, may produce a small portion of light that is scattered in a variety of directions. Light that is scattered but continues to have the same wavelength as the incident radiation will also have the same energy, a condition often referred to as Rayleigh or elastically scattered light. Incident radiation that is scattered during a change of vibrational state in molecules may be scattered with a different energy, and such scattered light may be called Raman scattered light. Such phenomena have been used in conjunction with spectroscopy to qualitatively and quantitatively study physical properties and processes, including identification of chemical properties, compositions, and structures of a sample.
A wave associated with electromagnetic radiation may be described by wavelength, the physical length of one complete oscillation, and by frequency of the wave, the number of oscillations per second that pass a point. If incident radiation is directed at a sample, the wavelength of the incident radiation may remain substantially unchanged in scattered radiation. Alternatively, if incident radiation is directed at a sample, the wavelength in the scattered radiation may acquire one or more different wavelengths than the incident wavelength. The energy differential between the incident radiation and the scattered radiation may be referred to as a Raman shift. Spectroscopic measurement of Raman scattered light seeks in part to measure the resulting wavelength of such scattered light.
Raman scattered light may occur at wavelengths shifted from the incident light by quanta of molecular vibrations. The phenomenon of Raman scattered light, therefore, is useful in spectroscopy applications for studying qualities and quantities of physical properties and processes, including identification of chemical properties, compositions, and structure in a sample. Currently, Raman shift spectroscopic analytical techniques are used for qualitative and quantitative studies of samples. If incident radiation is used to scatter light from a sample, and scattered radiation data is measured, the scattered radiation may provide one or more frequencies associated with the sample, as well as the intensities of those shifted frequencies. The frequencies may be used to identify the chemical composition of a sample. If, for example, intensities are plotted on a Y-axis, and frequency or frequencies are plotted on an X-axis, the frequency or frequencies may be expressed as a wave number, the reciprocal of the wavelength expressed in centimeters. The X-axis, showing frequency or frequencies, may be converted to a Raman shift in wave numbers, the measure of the difference between the observed wave number position of spectral bands, and the wave number of radiation appearing in the incident radiation.
While these principles and phenomena are known, until recently efforts to apply the principles and phenomena to qualitative and quantitative analyses of samples have not always resulted in uniform, predictable results, or in acceptable levels of precision and accuracy of Raman spectra. Because of instrumentation variabilities, inherent weakness of a Raman scattered signal, fluorescence, and other limitations associated with spectroscopy instruments, the goal of producing a standard Raman spectrum for use in sample analyses was, until recently, a challenge not achieved by apparatus and methods known in the art.
At least one problem that had to be overcome was the fact that spectroscopic measurements of Raman scattered light seeking to measure wavelength or intensities, or both, of scattered light, could be affected by the instrument, or spectroscopic system, itself. A number of components of an instrument may contribute individually and collectively to undesirable instrumentation variabilities that affect spectral data measured by the instrument. Raman scattered radiation from a sample may be observed, measured, and directed through an instrument by optics of a spectrometer, may be coded by a device such as an interferometer, and may be directed to one or more detectors to record Raman spectra. Any one, or all, of such components of a conventional spectrometer system induced or contributed to instrumentation variabilities that reduced or adversely affected the precision and accuracy of measurements of Raman scattered light.
In addition to fluorescence, spectral measurements of a source of incident radiation such as a laser, including semiconductor or diode lasers, will evidence other varying baseline components, artifactual or real, that preferably could be eliminated, suppressed, or compensated for to provide an accurate Raman spectrum for analytical purposes. In instrumentation designs preferred by users of Raman technology, semiconductor diode lasers would be the choice of incident radiation due to small and compact sizing, low heat dissipation, and high energy conversion efficiency. Use of semiconductor or diode lasers, while useful because of a number of important characteristics, also engender unique problems that, if solved, would advance Raman technology. However, at least one other problem associated with semiconductor diode lasers is the tendency for the output to change from one frequency to another during operation, commonly referred to as frequency drift. Frequency drift is generally related to temperature variations that may cause either slow frequency drifts or drastic frequency changes. Semiconductor diode lasers also are susceptible to mode hops when the laser switches output from one frequency to a new preferred frequency.
Some of the problems associated with frequency shifts were discussed as early as 1991 in Semiconductor Diode Lasers Volume I, edited by William Streifer and Michael Ettenberg, IEEE Press (1991), a work incorporated by reference into this document. In general, frequency shifts, or mode hops, are inherent in laser light, and can be eliminated only by redesigning the laser at excessive cost. Solutions for overcoming the effects of frequency shifts have included redesign of the internal cavity of lasers, designing what is known as an external cavity for lasers, and tuning a range of modes into a single mode. All of those solutions are achieved at considerable expense, and generally shorten the useful life of a semiconductor laser. A further problem related to diode lasers includes variations in output intensity that directly affect the measured Raman shift. Rather than eliminate the problem physically, which is expensive and limits the effective life of the laser, it would also be useful to compensate for the frequency shifts and intensity variabilities. Thus, it is at least an objective of the present invention to overcome problems associated with using excitation sources in the visible range of light, including, for example, removal of fluorescence and other common mode noise from acquired spectra.
Fortunately, in the Allen Patent, problems arising from instrumentation variabilities, including issues in connection with the use of semiconductor lasers, were overcome. The Allen Patent, incorporated by reference into this document as if fully set forth herein verbatim, discloses and claims an apparatus for measuring and applying instrumentation correction to produce a standard Raman spectrum. It would be novel and useful, however, to use the apparatus of the Allen Patent in a novel and unique way to provide a standard Raman spectrum by addressing primarily phenomena associated with sample interferences including common mode background interferences. To appreciate that contribution to Raman technology presented by the present invention, however, it is useful to review some perplexities of Raman phenomena.
Raman scattering is a comparatively weak effect when compared with Rayleigh or elastic scattering. Nevertheless, Raman scattering offers a significant opportunity for qualitative and quantitative studies of physical properties and processes, including identification of chemical compositions and structure in samples of physical matter. To appreciate these phenomena, as well as understand the problems solved by the present invention, it should be noted that depending on the compound comprising a sample, only about one scattered photon in 10.sup.6-8 tends to be Raman shifted. Because Raman scattering, therefore, is such a comparatively weak phenomenon, an instrument used to disperse radiation for measurement purposes should have minimal stray light and be able to substantially reject Rayleigh scattering; otherwise, a Raman shift may not be measurable.
As earlier described, Raman phenomena result in spectral information that is shifted relative to the excitation source, or source of incident radiation. Thus, any variations in the excitation source will result in a relative change, or shift, in spectral information. Spectrally shifted Raman information also is directly related to the intensity of the excitation source. A further complication arises from multiple lines in the frequency of the source of incident radiation that may cause shifted, multiple sets of spectra from a sample. Therefore, conventional Raman experimentation discloses that a source or sources of incident radiation that causes or cause excitation in a sample used in connection with a spectrograph should be substantially monochromatic, preferably providing a single frequency or wavelength. Recognition that the source of incident radiation requires a substantially monochromatic frequency has led to use of a variety of laser light sources as a source of incident radiation because of the substantially monochromatic frequency and high intensity of a laser. Gas lasers such as helium-neon, helium-cadmium, argon-ion, krypton-ion, as well as solid state lasers including Nd-YAG, and diode lasers, solid state tunable lasers, liquid dye lasers, and other lasers, have been used.
Preferably, a source of incident radiation would provide a substantially monochromatic frequency and radiation closer to the blue portion of the visible light spectrum providing short wavelength excitation because the Raman effect is enhanced by use of short wavelength excitation, and because of the enhanced quantum efficiency ("QE") of charged coupled detectors ("CCD's") in use today.
An undesirable result of incident radiation on a sample occurs if a sample generates red shifted radiation as part of a radiation absorption process, a phenomenon commonly referred to as fluorescence. Fluorescence occurs when absorbed radiation is lowered in frequency by internal molecular processes and emitted as radiation that is closer to the red end of the visible light spectrum. Fluorescence sometimes may be strong enough in comparison with the Raman shift to swamp, or substantially eliminate, the weaker Raman signal. Fluorescence is a major interference for samples using excitation wavelengths in the visible region of the light spectrum, and has therefore made use of blue and green excitation sources problematic. Using excitation sources in the far end of the red end of the light spectrum mitigates the fluorescence effect, however, particularly in connection with silicon detectors, but substantially restricts use of instrument components that tend to provide radiation far into the infrared ("IR") region of the light spectrum.
In one embodiment of the present invention, therefore, the apparatus collects a first spectral measurement from the incident beam and a first spectral measurement from the Raman beam. One or more frequency shifts are quantified, namely induced, identified and measured. Following the step of quantifying one or more frequency shifts, either induced or naturally occurring, the apparatus collects second spectral data from the incident beam and second spectral data from the Raman beam. Using the apparatus for measuring and applying instrumentation correction, spectral data modifications are applied to the resultant spectral data. As used in this document, the term "spectral data modifications" may include scaling, if necessary, where scaling includes one or more mathematical procedures for correcting ratio imbalances well known to those skilled in the art. The term "spectral data modifications" also includes a subtraction step in connection with the obtained spectral data. In addition, one or more integral transforms are applied to the respective spectral data. The resultant data then is deconvolved to produce the standard Raman spectrum of the sample.
In another embodiment of the present invention the standard Raman spectrum of a sample is obtained using a single spectrum. The apparatus collects a first spectral measurement set from the incident beam and Raman beam. The spectral measurement set frequency axis is shifted, and one or more shifted, spectral data set is collected. The alternative embodiment also applies one or more spectral data modifications to the spectral measurement set and to the one or more shifted spectral data sets to produce the standard Raman spectrum of the sample. One or more means for correcting spectral data, well known to those skilled in the art, may be used if necessary to flatten the baseline data to produce the standard Raman spectrum of the sample. The one or more means for correcting spectral data may include linearizing the spectral data; subtracting a spline, polynomial or other function; one or more pattern recognitions; Fourier filtering; or smoothing to reveal the details of the result, as well as one or more other methods well known in the art.
Earlier efforts to provide a fluorescence rejection technique were suggested in Effective Rejection of Fluorescence Interference in Raman Spectroscopy Using a Shifted Excitation Difference Technique, A. P. Shreve, N. J. Cherepy, and R. A. Mathies, 46 Applied Spectroscopy 707 (1992) ("Mathies Reference"), and Fluorescence Rejection in Raman Spectroscopy by Shifted-Spectra, Edge Detection, and FFT Filtering Techniques, P. A. Mosier-Boss, S. H. Lieberman, and R. Newberry, 49 Applied Spectroscopy 630 (1995) ("Lieberman Reference"). By those skilled in the art, it is recognized that fluorescence is a broad band spectral phenomena; small changes in frequency of the excitation source have little if any effect on the spectrum. This is not true of Raman phenomena, where small changes in excitation source frequency correspond to a similar change in the Raman shifted spectrum relative to the excitation source.
The Mathies Reference suggests obtaining a first spectra reading that includes a Raman reading and fluorescence reading, moving the laser to a shorter excitation frequency, and obtaining a second spectra that was subtracted from the first spectra. The author suggested that the resultant difference spectra would remove the broad band fluorescence spectrum, leaving only Raman shifted information. The resulting difference spectrum was fit in a non-linear least square process with sets of difference functions for each peak as determined by inspection of graphs of the different spectra, and a conventional spectrum was reconstructed using modeling techniques.
While the ideas of the Mathies Reference conceptually are useful, the apparatus and methods recommended included a number of problems. The shift in frequency of the excitation source must be known precisely, a requirement that led to use of a tunable laser. Tunable lasers are complex, bulky, and expensive. The output frequency of a tunable laser is assumed to remain stable, at a known frequency, during an entire integration time. If the frequency were to change during operation, the method proposed by the Mathies Reference would not work. Further, changes in output power of a tunable laser during sample integration time was unacceptable because of concern that the signal would scale with laser power, and cancellation of fluorescence would be incomplete. The Mathies Reference also poses a number of other problems associated with data interpretation, including the fact that a first approximation or estimate must be made for band centers, areas, and standard deviations of the difference spectra. An iterative non-linear least squares process then must be used to refine the first approximations to generate a best fit to the differential data. Further, the data analyses method of the Mathies Reference requires substantial and significant knowledge about the sample, including how many Raman bands are present in the spectrum. For each of the features, one must be able to make reasonable first approximations of the parameters fitted by an optimization routine, a process making it increasingly difficult to determine band areas and standard deviations for complex compounds. The Mathies Reference also makes use of a sample with only three Raman bands; more complex samples will exacerbate the limitations with this approach. Finally, the end result is not a measured result, but rather a modeled one, which limits its acceptance for certain applications such as forensic and FDA related applications.
The Lieberman Reference suggests use of a technique for using the shifted excitation Raman difference method of the Mathies Reference, but instead shifts either the spectrometer settings prior to collection of a second spectrum, or artificially shifts the digitized spectrum of the first acquisition. While this approach does not require a tunable laser beam source, it has all the limitations of the Mathies approach.
What is needed, therefore, is an apparatus in combination with a novel and useful spectra data analysis method that will overcome existing problems associated with implementing shifted excitation Raman difference spectroscopy. The need for such a method is evident because suppression of sample fluorescence, or other common mode interferences that are not effected by small changes in excitation source frequency, offers significant advantages. A method using an apparatus for adjusting spectral measurements to mitigate effects of fluorescence, and to produce a standard Raman spectrum, would allow measurements not only in the red or infrared spectrum of light, as is now commonly imposed by the nature of Raman technology, but also in the blue and green region. Maximum advantage could be achieved by using the green portion of the light spectrum, where the CCD (charge coupled devices) are most efficient. Inexpensive, unstabilized semiconductor diode lasers could be used. Modeling, or special understanding of differential data, would be unnecessary.
A number of problems must be solved to achieve the goal of providing a method for adjusting Raman spectra to produce a standard Raman spectrum using the concept of shifted excitation Raman differences to suppress or compensate for fluorescence and other common mode interferences that are not affected by small changes in the excitation source frequency. What is needed to solve the problems is a method for using an apparatus that is useful not only for measuring and applying instrumentation correction to produce a standard Raman spectrum, as provided in the Allen Patent, but also useful for inducing and monitoring a frequency shift at the user's direction. The apparatus should be capable of making one or more spectral measurements after occurrence of the frequency shift. The apparatus and method also should include ways to apply one or more arithmetic calculations to either a single spectral measurement, or to obtain a positive result in the nature of resulting spectral measurement data from subtracting a second spectral measurement from a first spectral measurement. The apparatus should allow application of one or more integral transforms to the resulting spectral measurement data to produce the standard Raman spectrum of the sample. The apparatus and method should be fully automated, relieving an operator of the apparatus from being either skilled in the art or possessing special skills, yet being capable of maintaining the quality of the data over a time period unmonitored or unattended by an operator of the system. In addition, high resolution Raman spectra should be achieved using an apparatus and method of operation of the apparatus that is easy to use, predictably accurate, easy to practice, and relatively cost effective.
One of many advantages of the new and useful present invention, therefore, is a method useful in adjusting spectral measurements occasioned by sample interferences to produce a standard Raman spectrum of the sample. The present invention, therefore, is useful in inducing and determining the occurrence of a frequency shift in the incident radiation and Raman radiation. The apparatus is capable of making one or more spectral measurements after occurrence of the frequency shift. The apparatus also is capable of making a first spectral measurement before the frequency shift and a second spectral measurement after the frequency shift. One or more arithmetic calculations, well known to those skilled in the art, are applied to the single spectral measurement to obtain a spectral measurement. Alternatively, the second spectral measurement may be subtracted from the first spectral measurement. The apparatus allows application of one or more integral transforms to the resulting spectral measurement data to produce the standard Raman spectrum of the sample. The apparatus and method are fully automated. In addition, the apparatus and method are easy to use, predictably accurate, easy to practice and are cost effective.
These advantages and other features of an apparatus and method for adjusting spectral measurements to produce a standard Raman spectrum will become apparent to those skilled in the art when read in conjunction with the accompanying following description, drawing figures, and appended claims.